Conserved region 3 of the adenovirus type 5 DNA-binding protein is important for interaction with single-stranded DNA.
The adenovirus-encoded single-stranded DNA-binding protein (DBP) functions in viral DNA replication and several aspects of RNA metabolism. Previous studies (G. A. M. Neale and G. R. Kitchingman, J. Biol. Chem. 264:3153-3159, 1989) have defined three highly conserved regions in the carboxy-terminal domain of the protein (amino acids 178 to 186, 322 to 330, and 464 to 475) that may be involved in the binding of the protein to single-stranded DNA. We examined the role of conserved region 3 (464 to 475) by constructing nine classes of point mutants with from one to four amino acid changes. The point mutants were tested for their ability to assist adeno-associated virus DNA replication. All nine differed from wild-type DBP; seven were essentially nonfunctional, whereas two had 55 and 145%, respectively, of the wild-type DBP helper activity. Three of the mutants were found to be temperature sensitive, with significantly greater helper activity at 33 degrees C than at 37 degrees C. All nine mutants produced essentially wild-type levels of protein. One monoclonal antibody against the DBP, termed 2/4, did not immunoprecipitate the mutant DBPs as well as wild-type DBP, indicating either that the antibody recognized sequences around CR3 or that the conformation of the protein around the epitope recognized by 2/4 had changed. Two of the three temperature-sensitive DBP mutants bound to single-stranded DNA-cellulose with the same affinity as wild-type DBP at 4 degrees C; the remaining mutants all showed reduced affinity. These results demonstrated that many of the residues within conserved region 3 of the DBP are important for interaction of the protein with nucleic acid.
Neale
G A
GA
Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
Kitchingman
G R
GR
D000260:Adenoviruses, Human / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005814:Genes, Viral; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids; D014162:Transfection; D015678:Viral Structural Proteins / Q000235:genetics*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D015345:Oligonucleotide Probes; D015678:Viral Structural Proteins